1. Field of the Invention
The present invention relates to the field of structural biology. More particularly, the methods of predicting pKa of a protein, the pH stability of a protein and electrostatic interactions of a protein.
2. Related Art
The solution behavior of a protein is a direct result of its chemical composition in coordination with the various conformational states it may adopt in the aqueous solvent. Enumerating these states and their free energy differences provide the information required to interpret stability, binding, allosteric effects, cooperative interactions, and function in terms of structure (Hilser et al., 1996; Hilser et al., 1997; Wooll et al., 2000; Hilser et al., 1998; Freire E., 1999; Pan et al., 2000; Freire et al., 1978; and Freire E., 1998).
Structural and energetic cataloging of states other than the “native” structure observed in crystallographic and NMR studies has proved elusive and exceedingly difficult to obtain by experiment due to the overwhelming free energy domination of the “native” state over partially folded conformers. (Kim et al., 1990; Kuwajima, 1989). But many observed protein phenomena (i.e., NMR studies on backbone dynamics; amide hydrogen exchange rates; mutational effects on binding, stability; and denaturant dependence of stability) are difficult to understand without postulating the existence and readily population of partially folded states.
Proton titration offers an ideal experimental technique for which to probe the local stability of various regions of a protein. Theoretical interpretation of proton binding curves are particularly informative because 1) protons bind non-homogeneously and to well defined sites, 2) the pKa of each binding site can be calculated directly from electrostatic theory if provided the structure (Klapper et al., 1986; Warwicker, J. 1986; Antosiewicz et al., 1994; Jayaram et al., 1989; Tanford et al., 1957; Matthew et al., 1986), and 3) the effect of proton binding on the free energy difference between the various conformational states of the ensemble is easily ascertained from linkage theory (Tanford, C 1969; Tanford, C. 1962).
A difficulty in using the above proton binding techniques is determining the ensemble of states populated at any solution pH and quantitating their structures and stabilities. The present invention is the first to address the role of partially folded states on the pH dependence of stability of proteins and how the electrostatic contribution to stability is tightly linked to structural dynamics.